Gene expression is composed of several major processes, including transcription, translation and protein processing. Among these processes, transcription not only dictates the precise copying of DNA into mRNA but also provides sophisticated mechanisms for the control of gene expression. There are a number of fundamental steps involved in transcription: promoter recognition and binding by transcription factors and RNA polymerase components, nascent RNA chain initiation, RNA transcript elongation, and RNA transcript termination (Uptain et al., Ann. Rev. Biochem. 66:117–172 (1997)). Promoters are an essential component for transcription, effecting transcription both quantitatively and qualitatively. A promoter contains numerous DNA motifs or cis-elements that can serve as recognition signals and binding sites for transcription factors. Working together with transcription factors, these cis-elements can function as architectural elements or anchoring points for achieving promoter geometry (Perez-Martin et al., Ann. Rev. Microbiol. 51:593–628 (1997)).
Numerous promoters have been isolated from a wide variety of organisms ranging from viruses to animals. They have become the subjects of intensive studies in efforts to characterize their molecular organization and the basic mechanisms regulating transcriptional control of gene expression. In recent years, a number of well-characterized promoters have been successfully adopted for use in the genetic transformation of plants. These promoters control transgene expression in transgenic plants and have been used in efforts to improve agronomic performance and to incorporate value-added features. However, in spite of the availability of these promoters, there is currently a shortage of promoters for use in genetic transformation research with plants. In most instances, use of existing plant promoters isolated from a specific species to effect transformation in a different species results in reduced promoter activity and/or altered patterns of gene expression, reflecting the variation of genetic background between different species (Ellis et al., EMBO J. 6:11–16 (1987); Miao et al., Plant Cell 3:11–22 (1991)). Recently, a constitutive actin gene promoter isolated from Arabidopsis (An et al., Plant J. 10:107–121 (1996)) failed to support desired levels of transgene expression in grape cells. To date, the promoter most commonly used to effect transformation in crop plants is the cauliflower mosaic virus 35S (CaMV 35S) promoter and its derivatives (Sanfacon, Can. J. Bot. 70:885–899 (1992)). The CaMV 35S promoter was originally isolated from a plant virus.
Successful genetic transformation of plants frequently requires the use of more than one promoter to adequately drive expression of multiple transgenes. For instance, at least three promoters are normally needed in order to express a selectable marker gene, a reporter marker gene and a target gene of interest. Multiple promoters are required because almost all the mRNAs in eukaryotes are monocistronic (single polypeptide-encoding transcript). Hence, expression of complex traits controlled by more than a single target gene in plants has been thought to require the use of additional promoters.
Recent studies have showed that foreign DNA integrated into the plant genome can be recognized by host factors and that the foreign DNA may be subsequently subjected to modifications that lead to transgene silencing. Mechanisms involved in this process include; DNA methylation, chromatin structural modification and post-transcriptional mRNA degradation (Kumpatla et al., TIBS 3:97–104 (1998)). In general, foreign DNA containing repeated sequences, including sequences homologous to host DNA, is more prone to gene silencing modifications (Selker, Cell 97:157–160 (1999)). Accordingly, the repeated use of the same promoter in transformation vector may increase the probability of gene silencing and unstable transgene expression in transgenic plants. As more transgenic crop plants are developed for release to the farmers, transgene silencing is likely to become a major concern. Hence, there is an urgent need to develop new promoters that will efficiently drive transgene expression, especially in transgenic plants.
Over the years, several strategies have been adopted for use to improve the performance of various promoters. These strategies can be classified into two categories, namely 1) modification of homologous promoters and 2) construction of heterologous promoters.
Modification of homologous promoters is accomplished by manipulating the enhancer region of a particular promoter in an effort to achieve higher transcriptional activity without altering existing expression patterns. Kay et al. (Science 236:1299–1302 (1987) first demonstrated that approximately ten-fold higher transcriptional activity was achieved by tandem duplication of 250 base pairs of the upstream enhancer region of the CaMV 35S promoter, as compared to the transcriptional activity of the natural promoter. Mitsuhara et al. (Plant Cell Physiol. 37:49–59 (1996)) further showed that other forms of tandem repeats of the upstream enhancer region of the CaMV 35S promoter were also capable of producing 10 to 50 fold higher levels of transgene expression in rice and tobacco without altering the constitutive expression pattern of the promoter.
Modification of promoters using heterologous enhancer sequences is also commonly practiced to achieve higher transcriptional activity and desired expression patterns. For example, a CaMV 35S promoter upstream enhancer fragment was fused to the nopaline synthase promoter (NOS) and the resulting fusion promoter reportedly increased the transcriptional activity, as compared to the weaker NOS promoter (Odell, et al. PMB 10:263–272 (1988)). The upstream enhancer regions of the CaMV 35S promoter and the octopine synthase promoter were used to fuse with the maize Adhl promoter to enhance transcription activity, while retaining the anaerobic regulation pattern of the Adhl promoter (Ellis et al. EMBO J.6:11–16 (1987) and 6:3203–3208 (1987)). The achievement of transcriptional enhancement by using heterologous enhancers is primarily attributable to the unique characteristics of enhancers, which could exert its functions to regulate transcriptional activity in an orientation- and position-independent fashion.